Design and optimization of multicolor panels

Holden T. Maecker

Outline

• Choosing fluorochrome combinations and filter sets

• Matching antibody specificities with fluorochromes

• Controls and standardization

Outline

• Choosing fluorochrome combinations and filter sets

• Choose bright fluorochromes

• Minimize spillover between detectors

“Bright” = good resolution sensitivity

W2

W1

D

WD(SI)Index Stain

Where D = difference between positive and negative peak medians, andW = 2 x rSD (robust standard deviation)

Various fluorochromes-stain index

Reagent Clone Filter Stain Index

PE RPA-T4 585/40 356.3

Alexa 647 RPA-T4 660/20 313.1

APC RPA-T4 660/20 279.2

PE-Cy7 RPA-T4 780/60 278.5

PE-Cy5 RPA-T4 695/40 222.1

PerCP-Cy5.5 Leu-3a 695/40 92.7

PE-Alexa 610 RPA-T4 610/20 80.4

Alexa 488 RPA-T4 530/30 75.4

FITC RPA-T4 530/30 68.9

PerCP Leu-3a 695/40 64.4

APC-Cy7 RPA-T4 7801/60 42.2

Alexa 700 RPA-T4 720/45 39.9

Pacific Blue RPA-T4 440/40 22.5

AmCyan RPA-T4 525/50 20.2

Spillover affects resolution sensitivity

FITC background contributes noise to PE measurement

www.bdbiosciences.com/spectra

Choices for 6-, 8-, 10-, and more colors

6-color 8-color 10-color AdditionalFITC or Alexa 488 FITC or Alexa 488 FITC or Alexa 488 FITC or Alexa 488

PE PE PE PE

PE-Texas Red or PE-Alexa 610

PE-Texas Red or PE-Alexa 610

PerCP-Cy5.5 PerCP-Cy5.5 PerCP-Cy5.5 PerCP-Cy5.5

PE-Cy7 PE-Cy7 PE-Cy7 PE-Cy7

APC or Alexa 647 APC or Alexa 647 APC or Alexa 647 APC or Alexa 647

Alexa 680 or 700 Alexa 680 or 700

APC-Cy7 APC-Cy7 APC-Cy7 APC-Cy7

AmCyan AmCyan AmCyan

Pacific Blue Pacific Blue Pacific Blue

Q-dot 655, 705…

Outline

• Choosing fluorochrome combinations and filter sets

• Matching antibody specificities with fluorochromes

• Controls and standardization

Fluorochrome selection considerations

• “Bright” antibodies go on “dim” fluorochromes

• Avoid spillover from bright cell populations into detectors requiring high sensitivity

• Take special care with tandem dyes

uncompensated compensated

FITC mean fluorescence PE mean fluorescence---------------------------- ----------------------------negative positive negative positive----------- ---------- ----------- ----------

uncompensated 123 3541 184 1648 compensated 123 3564 134 136

data spread

Data spread of fluorescent signals

HT Maecker, T Frey, LE Nomura, J Trotter: Selecting fluorochrome conjugates for maximum sensitivity. Cytometry A 2004, 62:169-73.

Spillover affects resolution sensitivity

Without CD45 AmCyan: With CD45 AmCyan:

CD19 FITC

Note that this is only an issue when the two markers (CD45 and CD19) are co-expressed on the same cell population.

Special requirements of tandem dyes

• Compensation requirements for tandem dye conjugates can vary, even between two experiments with the same antibody• Require experiment-specific compensation

• Certain tandem dye conjugates (APC-Cy7, PE-Cy7) can degrade with exposure to light, elevated temperature, and fixation• Minimize exposure to these conditions• Use BD Stabilizing Fixative for final fixation

False positives due to tandem degradation

A.

False positives inAPC channel reducedin absence of APC-Cy7

False positivesin PE channelremain

Gating scheme CD8 APC-Cy7+ cells CD4 PE-Cy7+ cells

B.

With CD8 APC-Cy7 and CD4 PE-Cy7:

Without CD8 APC-Cy7:

New tandems will be more stable

• APC-H7 as a replacement for APC-Cy7:

Comparison of Sample Stability

(in BD Stabilizing Fixative at RT)

0

50

100

150

200

250

0 1 2 4 6 8 24 48

Hours of light exposure

% S

pillo

ver

CD4 APC-H7

CD8 APC-H7

CD4 APC-Cy7

CD8 APC-Cy7

Outline

• Choosing fluorochrome combinations and filter sets

• Matching antibody specificities with fluorochromes

• Controls and standardization

Types of controls

• Instrument setup controls• PMT voltage settings• Compensation (per experiment)

• Gating controls• Isotype controls• Fluorescence-minus-one (FMO) controls

• Biological controls• Unstimulated samples• Healthy donors

Comparison of gating controls

Standardization using lyophilized reagents

• Lyophilization provides increased stability, even at room temperature or 37oC

• One batch of reagents can be used for an entire longitudinal study

• Pre-configured plates can avoid errors of reagent addition

• Complex experiments (multiple stimuli, multiple polychromatic staining cocktails) become easier

• Lyophilized cell controls can provide run-to-run standardization

Conclusions

• Polychromatic flow cytometry is not impossible

• Select fluorochromes for brightness and least spillover

• Optimize antibody panels by taking into account reagent brightness and data spread

• Stabilize longitudinal experiments with proper QC

• Some solutions that can help

• Lyophilized reagent plates

• Stabilizing fixative

• Beads for calibration and compensation

References

• Maecker, H. T., Frey, T., Nomura, L. E., and Trotter, J. (2004).Selecting fluorochrome conjugates for maximum sensitivity. Cytometry A 62, 169.

• Maecker, H. T., and Trotter, J. (2006).Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A 69, 1037.

Acknowledgements

• Laurel Nomura• Margaret Inokuma• Maria Suni• Smita Ghanekar• Daiva Gladding• Jack Dunne• Skip Maino

• Joe Trotter• Dennis Sasaki• Marina Gever